Thermostable purified endoglucanas from acidothermus cellulolyticus ATCC 43068

• US 5,275,944 A
• Filed: 01/27/1992
• Issued: 01/04/1994
• Est. Priority Date: 09/26/1989
• Status: Expired due to Term

• Current Assignee: Alliance For Sustainable Energy LLC
• Original Assignee: Midwest Research Institute
• Inventors: Grohmann, Karel, Himmel, Michael E., Adney, William S., Tucker, Melvin P.
• Primary Examiner(s): Naff, David M.
• Assistant Examiner(s): Meller, Michael V.
• Application Number:
  US07/826,089
• Time in Patent Office: 708 Days
• Field of Search: 435/209, 435/822, 435/814, 435/252.1
• US Class Current: 435/209
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: Y10S 435/814 Enzyme separation or purification
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales

Abstract

A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068). The cellulase is water soluble, possesses both C₁ and C_x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83° C. at pH'"'"'s from about 2 to about 9, and in inactivation temperature of about 110° C. at pH'"'"'s from about 2 to about 9.

Claims

1. A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus ATCC 43068, said cellulase is water soluble, possesses both C₁ and C_x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83°

C. at pH'"'"'s from about 2 to about 9, and an inactivation temperature of about 110°

C. at pH'"'"'s from about 2 to about 9.

2. The purified cellulase of claim 1, wherein the pH for the optimum temperature activity is from about 4 to about 8, and the pH for the inactivation temperature is from about 4 to about 8.

3. The purified cellulase of claim 1, wherein the pH for the optimum temperature activity is about 5, and the pH for the inactivation temperature is about 5.

4. A method for the production of the cellulase of claim 1, comprising culturing said bacterium in a nutrient medium therefor, filtering crude cellulase complex by ultrafiltration, separating unpurified high molecular weight and low molecular weight ultrafiltrate fractions by size exclusion chromatography, adding CsCl and a concentrated acetate buffer to said low molecular weight fraction, passing said low molecular weight fractions through an ion exchange tolumn equilibrated against 15 mM Bis-Tris pH 7 loading buffer, and recovering said low molecular weight cellulase fractions by using a shallow gradient of an increasing ionic strength buffer that increases linearly from 0 to about 50 mM of NaCl, until purified low molecular weight cellulase fractions are eluted off at about 25 mM.